Electrochemical denaturation and annealing of nucleic acid

ABSTRACT

A process is described for denaturing native double-stranded nucleic acid material into its individual strands in an electrochemical cell. The process disclosed is an electrical treatment of the nucleic acid with a voltage applied to the nucleic acid material by an electrode. The process may also employ a promoter compound such as methyl viologen to speed denaturation. The process may be used in the detection of nucleic acid by hybridizing with a labelled probe or in the amplification of DNA by a polymerase chain reaction or ligase chain reaction. A process is described for annealing complementary strands of nucleic acid by application of an electrical voltage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/617,675, filed on Apr. 1, 1996, U.S. Pat. No. 5,824,477, which is acontinuation-in-part application of U.S. patent application Ser. No.08/288,231, filed on Aug. 9, 1994, issued on Jun. 18, 1996 as U.S. Pat.No. 5,527,670, which is a continuation application of 08/030,138, filedon Mar. 12, 1993, now abandoned, which is a 371 of PCT/GB91/01563, filedon Sep. 12, 1991, the disclosure of each of which is incorporated hereinby reference in its entirety for all purposes. This application alsoclaims the priority dates of Sep. 12, 1990, Jun. 14, 1991, and Sep. 12,1991, the filing dates of the corresponding United Kingdom patentapplication 9019946.4, 9112911.4, and PCT application PCT/GB/01563,respectively, the disclosure of each of which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to processes for the treatment of nucleic acidmaterial in order to effect a complete or partial change fromdouble-stranded form to single-stranded form and to processes ofamplifying or detecting nucleic acids involving such denaturationprocesses.

Double-stranded DNA (deoxyribonucleic acid) and DNA/RNA (ribonucleicacid) and RNA/RNA complexes in the familiar double helical configurationare stable molecules that, in vitro, require aggressive conditions toseparate the complementary strands of the nucleic acid. Known methodsthat are commonly employed for strand separation require the use of hightemperatures of at least 60° Celsius and often 100° C. for extendedperiods of ten minutes or more or use an alkaline pH of 11 or higher.Other methods include the use of helicase enzymes, such as Rep proteinof E. coli, that can catalyze the unwinding of the DNA in an unknownway, or binding proteins such as 32-protein of E. coli phage T4 that actto stabilize the single-stranded form of DNA. The denaturedsingle-stranded DNA produced by the known processes of heat or alkali isused commonly for hybridization studies or is subjected to amplificationcycles.

2. Description of the Prior Art

U.S. Pat. No. 4,683,202 (Mullis et al., assigned to Cetus Corporation)discloses a process for amplifing and detecting a target nucleic acidsequence contained in a nucleic acid or mixture thereof by separatingthe complementary strands of the nucleic acid, hybridizing with specificoligonucleotide primers, extending the primers with a polymerase to formcomplementary primer extension products and then using those extensionproducts for the further synthesis of the desired nucleic acid sequenceby allowing hybridization with the specific oligonucleotide primers totake place again. The process can be carried out repetitively togenerate large quantities of the required nucleic acid sequence fromeven a single molecule of the starting material. Separation of thecomplementary strands of the nucleic acid is achieved preferably bythermal denaturation in successive cycles, since only the thermalprocess offers simple reversibility of the denaturation process toreform the double-stranded nucleic acid, in order to continue theamplification cycle. However, the need for thermal cycling of thereaction mixture limits the speed at which the multiplication processcan be carried out, owing to the slowness of typical heating and coolingsystems. It also requires the use of special heat resistant polymeraseenzymes from thermophilic organisms for the primer extension step, ifthe continuous addition of heat labile enzyme is to be avoided. Itlimits the design of new diagnostic formats that use the amplificationprocess because heat is difficult to apply in selective regions of adiagnostic device and it also can be destructive to the structure of theDNA itself because the phosphodiester bonds may be broken at hightemperatures leading to a collection of broken single strands. It isgenerally believed that the thermophilic polymerases in use today have alower fidelity, i.e., make more errors in copying DNA, than do enzymesfrom mesophiles. It is also the case that thermophilic enzymes, such asTAQ polymerase have a lower turnover number than heat labile enzymes,such as the Klenow polymerase from E. coli. In addition, the need toheat to high temperatures, usually 90° C. or higher to denature thenucleic acid leads to complications when small volumes are used as theevaporation of the liquid is difficult to control. These limitationshave so far placed some restrictions on the use of the Mullis et al.process in applications requiring very low reagent volumes to providereagent economy, in applications where the greatest accuracy of copy isrequired, such as in the Human Genome sequencing project and in theroutine diagnostics industry where reagent economy, the design of theassay format and the speed of the DNA denaturation/renaturation processare important.

Denaturation/renaturation cycles are also required in order to performthe so-called ligase chain reaction described in EP-A-0320308, in whichamplification is obtained by ligation of primers hybridized to templatesequences, rather than by extending them.

It is known that DNA has electrochemical properties. For example, N. L.Palacek, in “Electrochemical Behaviour of Biological Macromolecules,”Bioelectrochemisipy and Bioenergetics, 15 (1986), pp. 275-295, disclosesthe electrochemical reduction of adenine and cytosine in thermallydenatured single-stranded DNA at about −(minus) 1.5 V. on the surface ofa mercury electrode. This reduction process also requires a priorprotonation and therefore takes place at a pH below 7.0. The primaryreduction sites of adenine and cytosine form part of the hydrogen bondsin the Watson-Crick base pairs. Palacek was unable to demonstratereduction of adenine and cytosine in intact, native double-stranded DNAat the mercury electrode. Palacek has further demonstrated that, to avery limited extent, the DNA double helix is opened on the surface ofthe mercury electrode at a row range of potentials centered at −(minus)1.2 V. in a slow process involving an appreciable part of the DNAmolecule. This change in the helical structure of the DNA is thought tobe due to prolonged interaction with the electrode charged to certainpotentials and is not thought to b e a process involving electrontransfer to the DNA. No accumulation of single-stranded DNA in theworking solution was obtained and no practical utility for thephenomenon was suggested. Palacek also reports that the guanine residuesin DNA can be reduced at −(minus) 1.8 V. to dihydroguanine which can beoxidized back to guanine at around −(minus) 0.3 V. The reducible guaninedouble bond is not part of the hydrogen bonds in the Watson-Crick basepairs and this electrochemical process involving guanine does not affectthe structure of the DNA double helix.

In an earlier paper, R. Jelen and E. Palacek (“NucleotideSequence-Dependent Opening of Double-Stranded DNA at an ElectricallyCharged Surface,”Gen. Physiol. Biophys., 4 (1985), pp. 219-237),describe in more detail the opening of the DNA double helix on prolongedcontact of the DNA molecules with the surface of a mercury electrode.The mechanism of opening of the helix is postulated to be anchoring ofthe polynucleotide chain via the hydrophobic bases to the electrodesurface after which the negatively charged phosphate residues of the DNAare strongly repelled from the electrode surface at an applied potentialclose to −(minus) 1.2 V, the strand separation being brought about as aresult of the electric field provided by the cathode. There is nodisclosure of separating the strands of the DNA double helix while theDNA is in solution (rather than adsorbed onto the electrode) and thereis no disclosure of useful amounts of single strand DNA in solution.Furthermore, there is no disclosure that the nucleotide base sequence ofthe DNA on the electrode is accessible from solution. The basesthemselves are tightly bound to the mercury surface. A mercury electrodeis a complex system and the electrode can only be operated in theresearch laboratory with trained technical staff.

H. W. Nurnberg, in “Applications of Advanced Voltammetric Methods inElectrochemistry,” Bioelectrochemistry, Plenum Inc. (New York), 1983,pp. 183-225, discloses partial helix opening of adsorbed regions ofnative DNA to a mercury electrode surface to form a so-called ladderstructure. However, the DNA is effectively inseparably bound to oradsorbed onto the electrode surface. In this condition,. it is believedthat the denatured DNA is of no use for any subsequent process ofamplification or hybridization analysis. To be of any use, the denaturedDNA must be accessible to subsequent processes and this is convenientlyachieved if the single-stranded DNA is available in free solution or isassociated with the electrode in some way but remains accessible tofurther processes. Nurnberg has not demonstrated the ability of themercury electrode to provide useful quantities of single-stranded DNA.

V. Brabec and K. Niki (“Raman scattering from nucleic acids adsorbed ata silver electrode” in Biophysical Chemistry, 23 (1985), pp. 63-70) haveprovided a useful summary of the differing views from several workers onDNA denaturation at the surface of both mercury and graphite electrodescharged to negative potentials. There has emerged a consensus amongstthe research workers in this field that the denaturation process onlytakes place in DNA that is strongly adsorbed to the electrode surfaceand only over prolonged periods of treatment with the appropriatenegative voltage, a positive voltage having no effect on the doublehelix.

Brabec and Palacek (J. Electroanal. Chem., 88 (1978), pp. 373-385)disclose that sonicated DNA damaged by gamma radiation is transientlypartially denatured on the surface of a mercury pool electrode, theprocess being detectable by reacting the single-stranded products withformaldehyde so as to accumulate methylated DNA products in solution.Intact DNA did not show any observable denaturation.

SUMMARY OF INVENTION

The present invention provides a process for denaturing double-strandednucleic acid which comprises operating on solution containing nucleicacid with an electrode under conditions such as to convert a substantialportion of said nucleic acid to a wholly or partially single-strandedform.

It has been found that it is possible to produce the denaturation ofundamaged, i.e., non-irradiated, DNA at ambient temperature by applyinga suitable voltage to a solution in which the DNA is present undersuitable conditions.

The mechanism for the process has not yet been fully elucidated. It isbelieved that the process is one in which the electric field at theelectrode surface produces the denaturation of the double helix when theDNA is in close proximity to the electrode, but not bound irreversiblyto it.

The process is found to be readily reversible. In polymerase chainreaction processes exemplified hereafter, it is shown that the denaturedDNA produced by the denaturing process of the invention is immediatelyin a suitable state for primer hybridization and extension. On a largerscale, it is found that samples of denatured DNA produced using anegative voltage electrode can be caused or encouraged to renature byreversal of the voltage or by incubation at a higher temperature toencourage reannealing.

Preferably, according to the invention, the single-stranded nucleic acidproduced is free from the electrode, e.g., in solution. However, thenucleic acid may be immobilized on the electrode in double orsingle-stranded form prior to the application of the electrodepotential, e.g., attached by the end or a small portion intermediate theends of the nucleic acid chain, so as to leave substantial segments ofthe nucleic acid molecules freely pendant from the electrode surfacebefore and after denaturation.

Preferably, a potential of from −0.2 to −3 V, (more preferably from −0.5to −1.5 V) is applied to said working electrode with respect to thesolution. More preferably still, the voltage is from −0.8 to −1.1 V,e.g., about −1.0 V.

Working electrode voltages are given throughout as if measured or asactually measured relative to a calomel reference electrode (BDH No.309.1030.02).

In addition to said electrode and a counter-electrode, a referenceelectrode may be contacted with said solution and a voltage may beapplied between said electrode and said counter-electrode so as toachieve a desired controlled voltage between said electrode and saidreference electrode. The electrodes may be connected by a potentiostatcircuit as is known in the electrochemical art.

The ionic strength of said solution is preferably no more than 250 mM,more preferably no more than 100 mM. As it has been found that the rateof denaturation increases as the ionic strength is decreased, the saidionic strength is still more preferably no more than 50 mM, e.g., nomore than 25 mM or even no more than 5 mM. Generally, the lower theionic strength, the more rapid is the denaturation. However, incalculating ionic strength for these purposes it may be appropriate toignore the contribution to ionic strength of any component which acts asa promoter as described below.

The solution may contain or the electrode may have on its surface apromoter compound which assists said denaturation.

Although the invented process can take place in a solution containingonly the electrode and the nucleic acid dissolved in water optionallycontaining a suitable buffer, the process can be facilitated by thepresence in the solution containing the nucleic acid of such a promotercompound.

The compound may act as a promoter serving either to destabilize thedouble-stranded nucleic acid, for instance by intercalation into thedouble helix, or to stabilize the single-stranded form, or else tofacilitate interaction between the electrode surface and the nucleicacid. By way of analogy, it has been found that 4,4′-bipyridyl promotesthe reduction of cytochrome C at a gold electrode, even though bipyridylis, in itself, electroinactive at the voltages used. (M. J. Eddowes andH. A. Hill, J. Chem. Soc. Chem. Commun. (1977), 771.)

The promoter may be any inorganic or organic molecule which increasesthe rate or extent of denaturation of the DNA double helix. It should besoluble in the chosen solvent. It preferably does not affect orinterfere with DNA or other materials (such as enzymes oroligonucleotide probes) which may be present in the solution.Alternatively, the promoter may be immobilized to or included in thematerial from which the electrode is constructed.

In experiments, it has been found that the promoter may be a watersoluble compound of the bipyridyl series, especially a viologen such asmethyl viologen or a salt thereof.

It is believed that the positively-charged viologen molecules interactbetween the negatively-charged DNA and the negatively-charged cathode toreduce electrostatic repulsion therebetween and, hence, to promote theapproach of the DNA to the electrode surface where the electrical fieldis at its strongest. Accordingly, we prefer to employ as promotercompounds having spaced positively charged centers, e.g., bipolarpositively charged compounds. Preferably, the spacing between thepositively charged centers is similar to that in viologens. Othersuitable viologens include ethyl viologen, isopropyl viologen and benzylviologen.

The process may be carried out in an electrochemical cell of the typedescribed by C. J. Stanley, M. Cardosi and A. P. F. Turner,“Amperometric Enzyme Amplified Immunoassays,” J. Immunol. Meth., 112(1988), pp. 153-161, in which there is a working electrode, a counterelectrode and, optionally, a reference electrode. The working electrodeat or by which the denaturing nucleic acid is effected may be of anyconvenient material, e.g., a noble metal such as gold or platinum, or aglassy carbon electrode.

The electrode may be a so-called “modified electrode,” in which thedenaturing is promoted by a compound coated onto, or adsorbed onto, orincorporated into the structure of the electrode which is otherwise ofan inert but conducting material. In an alternative electrochemical cellconfiguration, the working, counter and reference electrodes may beformed on a single surface, e.g., a flat surface by any printing methodsuch as thick film screen printing, ink jet printing, or by using aphoto-resist followed by etching. It is also possible that the workingand reference electrodes can be combined on the flat surface leading toa two-electrode configuration where the reference also acts as thecounter. Alternatively, the electrodes may be formed on the insidesurface of a well which is adapted to hold liquid. Such a well could bethe well-known 96 well or Microtitre plate, it may also be a test tubeor other vessel. Electrode arrays in Microtitre plates or other moldedor thermoformed plastic materials may be provided for multiple nucleicacid denaturation experiments.

The strand separation may be carried out in an aqueous medium or in amixture of water with an organic solvent such as dimethylformamide. Theuse of polar solvents other than water or non-polar solvents is alsoacceptable, but is not preferred. The process may be carried out atambient temperatures or, if desired, temperatures up to adjacent thepre-melting temperature of the nucleic acid. The process may be carriedout at pHs of from 3 to 10, conveniently about 7. Generally, more rapiddenaturation is obtained at lower pH. For some purposes, therefore, a pHsomewhat below neutral, e.g., about pH 5.5, may be preferred. Thenucleic acid may be dissolved in an aqueous solution containing a bufferwhose nature and ionic strength are such as not to interfere with thestrand separation process.

The denaturing process according to the invention may be incorporated asa step in a number of more complex processes, e.g., procedures involvingthe analysis and/or the amplification of nucleic acid. Some examples ofsuch applications are described below.

The invention includes a process for detecting the presence or absenceof a predetermined nucleic acid sequence in a sample which comprises:denaturing a sample double-stranded nucleic acid by means of a voltageapplied to the sample in a solution by means of an electrode;hybridizing the denatured nucleic acid with an oligonucleotide probe forthe sequence; and determining whether the said hybridization hasoccurred.

Thus, the invented process has application in DNA and RNA hybridizationwhere a specific gene sequence is to be identified, e.g., specific to aparticular organism or specific to a particular hereditary disease ofwhich sickle cell anemia is an example. To detect a specific sequence,it is first necessary to prepare a sample of DNA, preferably of purifiedDNA, means for which are known, which is in native double-stranded form.It is then necessary to convert the double-stranded DNA tosingle-stranded form before a hybridization step with a labellednucleotide probe which has a complementary sequence to the DNA samplecan take place. The denaturation process of the invention can be usedfor this purpose in a preferred manner by carrying out the followingsteps:

denaturing a sample of DNA by applying a voltage by means of anelectrode to the sample DNA with optionally a promoter in solution orbound to or part of the structure of the electrode;

hybridizing the denatured DNA with a directly labelled or indirectlylabelled nucleotide probe complementary to the sequence of interest; and

determining whether the hybridization has occurred, which determinationmay be by detecting the presence of the probe, the probe being directlyradio-labelled, fluorescent labelled, chemiluminescent labelled orenzyme labelled or being an indirectly labelled probe which carriesbiotin, for example, to which a labelled avidin or avidin-type moleculecan be bound later.

In a typical DNA probe assay, it is customary to immobilize the sampleDNA to a membrane surface which may be composed of neutral or chargednylon or nitrocellulose. The immobilization is achieved by chargeinteractions or by baking the membrane-containing DNA in an oven. Thesample DNA can be heated to high temperature to ensure conversion tosingle-stranded form before binding to the membrane or it can be treatedwith alkali once on the membrane to ensure conversion to thesingle-stranded form. The disadvantages of the present methods are:

heating to high temperatures to create single-stranded DNA can causedamage to the sample DNA itself; and

the use of alkali requires an additional step of neutralization beforehybridization with the labelled probe can take place.

One improved method for carrying out DNA probe hybridization assays isthe so-called “sandwich” technique where a specific oligonucleotide isimmobilized on a surface. The surface having the specificoligonucleotide thereon is then hybridized with a solution containingthe target DNA in a single-stranded form, after which a second labelledoligonucleotide is then added which also hybridizes to the target DNA.The surface is then washed to remove unbound labelled oligonucleotide,after which any label which has become bound to target DNA on thesurface can be detected later.

This procedure can be simplified by using the denaturing process of theinvention to denature the double-stranded DNA into the requiredsingle-stranded DNA. The working electrode, counter electrode andoptionally a reference electrode and/or a promoter can be incorporatedinto a test tube or a well in which the DNA probe assay is to be carriedout. The DNA sample and oligonucleotide probes can then be added and thevoltage applied to denature the DNA. The resulting single-stranded DNAis hybridized with the specific oligonucleotide immobilized on thesurf-ace after the remaining stages of a sandwich assay are carried out.All the above steps can take place without a need for high temperaturesor addition of alkali reagents as in the conventional process.

The electrochemical denaturation of DNA can be used in the amplificationof nucleic acids, e.g., in a polymerase chain reaction or ligase chainreaction amplification procedure. Thus, the present invention provides aprocess for replicating a nucleic acid which comprises: separating thestrands of a sample double-stranded nucleic acid in solution under theinfluence of an electrical voltage applied to the solution from anelectrode; hybridizing the separated strands of the nucleic acid with atleast one oligonucleotide primer that hybridizes with at least one ofthe strands of the denatured nucleic acid; synthesizing an extensionproduct of the or each primer which is sufficiently complementary to therespective strand of the nucleic acid to hybridize therewith; andseparating the or each extension product from the nucleic acid strandwith which it is hybridized to obtain the extension product.

In such a polymerase mediated replication procedure, e.g., a polymerasechain reaction procedure, it may not be necessary in all cases to carryout denaturation to the point of producing wholly single-strandedmolecules of nucleic acid. It may be sufficient to produce a sufficientlocal and/or temporary weakening or separation of the double helix inthe primary hybridization site to allow the primer to bind to itstarget. Once the primer is in position on a first of the target strands,rehybridization of the target strands in the primer region will beprevented and the other target strand may be progressively displaced byextension of the primer or by further temporary weakening or separationprocesses.

Preferably, the said amplification process further comprises repeatingthe procedure defined above cyclicly, e.g., for more than 10 cycles,e.g., up to 20 or 30 cycles. In the amplification process, thehybridization step is preferably carried out using two primers which arecomplementary to different strands of the nucleic acid.

The denaturation to obtain the extension products as well as theoriginal denaturing of the target nucleic acid is preferably carried outby applying to the solution of the nucleic acid a voltage from anelectrode.

The process may be a standard or classical PCR process for amplifying atleast one specific nucleic acid sequence contained in a nucleic acid ora mixture of nucleic acids wherein each nucleic acid consists of twoseparate complementary strands, of equal or unequal length, whichprocess comprises:

(a) treating the strands with two oligonucleotide primers, for eachdifferent specific sequence being applied, under conditions such thatfor each different sequence being amplified, an extension product ofeach primer is synthesized which is complementary to each nucleic acidstrand, wherein said primers are selected so as to be substantiallycomplementary to different strands of each specific sequence such thatthe extension product synthesized from one primer, when it is separatedfrom its complement, can serve as a template for synthesis of theextension product of the other primer;

(b) separating the primer extension products from the templates on whichthey were synthesized to produce single-stranded molecules by applying avoltage from an electrode to the reaction mixture; and

(c) treating the single-stranded molecules generated from step (b) withthe primers of step (a) under conditions such that a primer extensionproduct is synthesized using each of the single strands produced in step(b) as a template.

Alternatively, the process may be any variant of the classical orstandard PCR process, e.g., the so-called “inverted” or “inverse” PCRprocess or the “anchored” PCR process.

The invention therefore includes an amplification process as describedabove in which a primer is hybridized to a circular nucleic acid and isextended to form a duplex which is denatured by the denaturing processof the invention, the amplification process optionally being repeatedthrough one or more additional cycles.

More generally, the invention includes a process for amplifying a targetsequence of nucleic acid comprising hybridization, amplification anddenaturation of nucleic acid, e.g., cycles of hybridizing anddenaturing, wherein said denaturation is produced by operating on asolution containing said nucleic acid with an electrode.

The process of the invention is applicable to the ligase chain reaction.Accordingly, the invention includes a process for amplifying a targetnucleic acid comprising the steps of:

(a) providing nucleic acid of a sample as single-stranded nucleic acid;

(b) providing in the sample at least four nucleic acid probes wherein:

i) the first and second of said probes are primary probes, and the thirdand fourth of said probes are secondary nucleic acid probes; ii) thefirst probe is a single strand capable of hybridizing to a first segmentof a primary strand of the target nucleic acid; iii) the second probe isa single strand capable of hybridizing to a second segment of saidprimary strand of the target nucleic acid; iv) the 5′ end of the firstsegment of said primary strand of the target is positioned relative tothe 3′ end of the second segment of said primary strand of the target toenable joining of the 3′ end of the first probe to the 5′ end of thesecond probe, when said probes are hybridized to said primary strand ofsaid target nucleic acid; v) the third probe is capable of hybridizingto the first probe; and vi) the fourth probe is capable of hybridizingto the second probe; and

(c) repeatedly or continuously: i) hybridizing said probes with nucleicacid in said sample; ii) ligating hybridized probes to form reorganizedfused probe sequences; and iii) denaturing DNA in said sample byapplying a voltage from an electrode to the reaction mixture.

In all of the amplification procedures described above, the denaturationof the DNA to allow subsequent hybridization with the primers can becarried out by the application of an appropriate potential to theelectrode. The process may be carried out stepwise involving successivecycles of denaturation or renaturation as in the existing thermalmethods of PCR and LCR, but it is also possible for it to be carried outcontinuously since the process of chain extension or ligation by theenzyme and subsequent strand separation by the electrochemical processcan continue in the same reaction as nucleic acid molecules insingle-stranded form will be free to hybridize with primers once theyleave the denaturing influence of the electrode. Thus, provided that theprimer will hybridize with the DNA an extension or ligation product willbe synthesized. The electrochemical DNA amplification technique can beused analytically to detect and analyze a very small sample of DNA,e.g., a single copy gene in an animal or a single cell of a bacterium.

The invention includes a kit for use in a process of detecting thepresence or absence of a predetermined nucleic acid sequence in a samplewhich kit comprises, an electrode, a counter electrode and optionally areference electrode, an oligonucleotide probe for said sequence. Theprobe may be labelled in any of the ways discussed above.

The invention also includes a kit for use in a process of nucleic acidamplification comprising an electrode, a counter electrode andoptionally a reference electrode, and at least one primer for use in aPCR procedure, or at least one primer for use in an LCR procedure,and/or polymerase or a ligase, and/or nucleotides suitable for use in aPCR process.

Preferably, such kits include a cell containing the electrodes.Preferably the kits include a suitable buffer for use in the detectionor amplification procedure.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the followingdrawings and examples.

FIG. 1 is a diagram of an electrochemical cell used for denaturation ofDNA;

FIG. 2 is a drawing of an electrophoresis gel showing the movement ofsingle and double-stranded DNA;

FIG. 3 is a diagram of an electrophoresis gel showing the electricaldenaturation of calf thymus DNA in the absence of any promoter such asmethyl viologen;

FIG. 4 is a diagram of an electrophoresis gel showing the renaturationof denatured calf thymus DNA;

FIG. 5 is a drawing of an electrophoresis gel showing the time course ofthe thermal denaturation of linear double stranded DNA from thebacteriophage M13;

FIG. 6 is a drawing of an electrophoresis gel showing the time course ofthe electrical denaturation of linear M13 DNA;

FIG. 7 is a drawing of an electrophoresis gel showing a comparison of athermally-amplified segment of M13 with an equivalent electricallyamplified segment of M13 in the presence of methyl viologen;

FIG. 8 is a drawing of an electrophoresis gel showing a comparison of athermally-amplified segment of M13 with an equivalent electricallyamplified segment in the absence of methyl viologen;

FIG. 9 is a drawing of an electrophoresis gel showing a fragment of“bluescript” DNA amplified using electrical PCR in the presence ofmethyl viologen; and

FIG. 10 is a drawing of an electrophoresis gel showing amplifiedfragments of two “bluescript” DNA's produced by electrical PCR in theabsence of methyl viologen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In FIG. 1, there is shown a cell structure 10 comprising a workingcompartment 12 in which there is a body of DNA-containing solution, aworking electrode 14, a counter electrode 16, a FiVac seal 19, a Kwikfit adaptor 21 and magnetic stirrer 18. A reference electrode 20 in aseparate side arm is connected via a “luggin” capillary 23 to thesolution in the sample 12. The working electrode, counter electrode andreference electrode are connected together in a potentiostat arrangementso that a constant voltage is maintained between the working electrode14 and the reference electrode 20. Such potentiostat arrangements arewell known (see for example “Instrumental Methods in Electrochemistry”by the Southampton Electrochemistry Group, 1985, John Wiley and Sons, p.19).

The electrode 14 is a circular glassy carbon rod of diameter 0.5 cm,narrowing to 0.25 cm at a height of 10 mM, and having an overall lengthof 9 cm inside a Teflon® sleeve of outside diameter 0.8 cm (supplied byOxford Electrodes, 18 Alexander Place, Abingdon, Oxon), and thereference electrode 16 is a 2 mm pin calomel (supplied by BDH No.309/1030/02). The counter electrode is supported by a wire which issoldered to a brass sleeve 25 above the adaptor and passes down andexits the Teflon® sleeve 20 mm from the base Xfothe working electrode.The wire attached to a cylindrical platinum mesh counter electrodesupplied by Oxford Electrodes which annularly surrounds the workingelectrode.

EXAMPLE 1

In this example of DNA denaturation, the two methods of thermal andelectrical denaturation have been compared. To achieve electricaldenaturation, 1.60 ml of solution of methyl viologen dichloride at 1mg/ml in distilled water (adjusted to pH 7 by titration with 0.1 Msodium hydroxide) was added to the working compartment of theelectrochemical cell described above. The reference arm of the cell inwhich the reference electrode 20 resides contained 0.4 ml of thissolution. A sample of 120 μm of a stock solution of calf thymus DNA(Sigma Chemical Company catalog number: D4522, average fragment size5,000 bases) at 1 mg/ml in distilled water (the pH was not adjusted) wasadded to the working chamber of the electrochemical cell to give a 70μg/ml final DNA concentration. The total ionic strength of the solutionwas calculated to be approximate 5 mM. A voltage of −(minus) 1.0 V wasapplied between the working electrode and the reference electrode.

The electrochemical cell was left for 16 hours at room temperature (22°C.) with continuous gentle stirring. On applying the potential to theworking electrode 14, the blue color of reduced methyl viologen wasobserved in the immediate vicinity of the working electrode. A 100 μlsample from the working compartment of the electrochemical cell wastaken at the end of the experiment and prepared for gel electrophoresisanalysis by mixing with 20 μl of gel loading buffer which was the sameas is described below for the gel itself which also contained 0.25%(w/v) bromophenol blue (BDH Indicators 200170), 0.25% (w/v) xylenecyanol (Sigma X2751), and 30% (v/v) glycerol (BDH AnalaR 100118). Themixtures were stored on ice prior to loading 10 μl samples in the wellscast in an agarose electrophoresis gel.

For the thermal denaturation experiment, the DNA solution at 1 mg/ml indistilled water (the pH was not adjusted) was heated to 100° C. for 10minutes in a boiling water bath. The tube containing the thermallydenatured DNA was then removed from the water bath and placedimmediately into a beaker containing an ice/water mix to ensure rapidcooling to prevent renaturation of the sample back to thedouble-stranded form. A 100 μl sample of the thermally denatured DNAsolution was prepared for gel electrophoresis by mixture with 20 μl ofgel loading buffer which contained 0.25% (w/v) bromophenol blue, 0.25%(w/v) xylene cyanol and 30% (w/v) glycerol.

Native intact calf thymus DNA was also prepared for gel electrophoresisby mixing 100 μl of the starting solution of DNA (before thermal ordenaturation) with 20 μl of gel loading buffer and stored on ice untilrequired.

The gel (a section of which is shown in FIG. 2) had a number of wells 30into which the samples could be loaded, and 10 μl samples were placedinto individual wells. The gel had a total volume of 50 ml and was 10 cmwide and 75 cm long; it was 0.5% (w/v) agarose in 0.089 M tris buffer pH8.0 containing 0.1 M borate and 0.01 M sodium EDTA. The gel was run for85 minutes at an applied constant voltage of 55 volts using a powersupply from Pharmacia No. 500/400. The gel was then removed from theelectrophoresis apparatus and stained by addition of 0.75 M of ethidiumbromide (Pharmacia No. 1840-501, lot 9503860E) at 20 μg/ml in distilledwater. After staining for 30 minutes, the gel was washed in distilledwater. The stained gel was trans-illuminated with ultraviolet light andthen photographed with a Polaroid® instant camera system using a redfilter to reduce background from the UV source.

The gel shown in FIG. 1 has samples A, B and C. Sample A was thestarting material used in the test (calf thymus DNA). Sample B was asample of calf thymus DNA which had been electrically denaturedaccording to the invention and sample C was a sample of thermallydenatured DNA. The DNA stain ethidium bromide becomes fluorescent whenit intercalates into the double helical structure of intact native DNA.Hence, it can be used to identify the double-stranded DNA in FIG. 1. Asthe DNA is denatured, it becomes progressively single-stranded and theefficiency of staining with ethidium bromide decreases. However, thereis still some residual staining of single-stranded calf thymus DNA,probably because there is still some ordering of the bases or even someregions which remain double-stranded to which ethidium bromide can bind.Therefore, the stain is still useful in detecting denatured DNA as wellas intact DNA. In FIG. 1 it will be noted that the samples B and C areof much higher mobility than sample A through the gel indicating thatthe DNA after thermal denaturation and after electrical reduction hassimilar physical characteristics, showing a denaturation to the singlestranded form which runs faster in this gel system. Similar results havebeen obtained using gold and platinum working electrodes.

EXAMPLE 2

Example 1 was repeated as before, but DNA samples were taken after 15minutes, 3 hours and 22 hours treatment in the electrochemical cell inorder to provide a time course of the denaturation of the DNA. Duringsampling from the cell, the potentiostat was switched to a dummy cellrepresented by a resistor. A gradual progressive denaturation of the DNAinto the faster migrating form on the gel was observed. The gel patternafter 15 minutes (not shown) is interpreted to represent a mixture ofpartially and fully denatured DNA but no evidence of wholly native DNAwas seen and, in later samples, the proportion of fully denatured DNAcontinued to increase.

EXAMPLE 3

In a number of runs at varying promoter concentrations and at a fixedDNA concentration of 20 μg/ml in the electrochemical cell and at anionic strength of 15 mM to 150 mM the rate of DNA denaturation was foundto be greatly accelerated by increasing the promoter concentration from3 to 30 mg/ml. At the higher concentration, denaturation of the DNA wasalmost complete in 15 minutes only. In this example, the overwhelminglypredominant source of ionic strength is the promoter itself.

EXAMPLE 4

At a fixed promoter concentration of 3 mg/ml and at an ionic strength of15 mM (derived essentially from the promoter) the rate of denaturationat various DNA concentrations was assessed. The results show that at 4μg/ml DNA the denaturation time was reduced to under 0.5 hours (asassessed by the disappearance of the double-stranded DNA band on theelectrophoresis gel).

The foregoing examples demonstrate that the ratio of DNA to the promotermethyl viologen affects the rapidity and completeness of thedenaturation of the DNA. These examples were carried out at low ionicstrength in distilled water. Repeating these examples at higher ionicstrength, i.e., 10 to 250 mM NaCl, lengthened the denaturation time ofthe DNA in solution by electrical means. This is attributed to theadditional stabilization that salt provides to the double helicalstructure of DNA.

The foregoing examples have also been repeated following exactly theprocedure described above for calf thymus DNA with other whole genomicDNA samples from salmon testes DNA (Sigma No. D1626) and with humanplacental DNA (Sigma No. D7011). Exactly the same results were obtainedon the agarose electrophoresis gel with these alternative DNA sources.

EXAMPLE 5

FIG. 3 shows the results of an electrical DNA denaturation carried outaccording to the methods described above but without the inclusion ofthe methyl viologen promoter. The ionic strength was less than 1 mM andthe calf thymus DNA concentration was 70 μg/ml. The electrochemical cellwas left for a total of 20 hours at ambient temperature (22° C.) withcontinuous gentle stirring at a potential of −(minus) 1.0 V on theworking electrode. Samples were taken from the cell at 1 hour, 3 hoursand 4 hours, as well as at the end of the procedure. During the samplingof the DNA solution from the cell, the potentiostat was switched to adummy cell represented by a resistor in order to avoid current surges.The gel in FIG. 3 shows that denaturation of the calf thymus DNA to thesingle-stranded form does indeed occur in the absence of promoter, butthe rate of the process was much slower than that observed with methylviologen present. Track A is the starting material, Track B is after 1hour, Track C after 3 hours, Track D after 4 hours and Track E after atotal of 20 hours at −(minus) 1 V. Some limited denaturation of the DNAwas observed within 1 hour, but considerable amounts of intact doublestranded DNA were still visible after 4 hours and only disappeared afterovernight incubation.

EXAMPLE 6

FIG. 4 shows the results from a renaturation procedure in whichelectrically denatured DNA has been treated under the appropriatethermal and electrical condition to recover the original double-strandedDNA. To an electrochemical cell of the type shown in FIG. 1, 2.45 ml ofdistilled water was added to the working compartment and 0.2 ml ofdistilled water was added to the reference compartment. To the workingcompartment were added 250 /μl of calf thymus DNA at 1 mg/ml indistilled water (pH not adjusted) and 100 μl of a solution of methylviologen at 100 mg/ml in distilled water (pH adjusted to pH 7 with 0.1 Msodium hydroxide). The final DNA concentration in the solution was 90μg/ml and the promoter 3.5 mg/ml. The ionic strength was approximately15 mM derived essentially from the promoter. After gentle stirring ofthe contents of the cell with the magnetic stirring bar, a 100 μl samplewas taken and stored on ice or frozen at −(minus) 20° C. The electrodeswere positioned in the cell, as illustrated in FIG. 1, and a voltage of−(minus) 1 V. was applied to the working electrode. The contents of thecell were stirred gently with the magnetic stirring bar and theconversion of the DNA to the single-stranded form continued for 90minutes at ambient temperature (22° C.). After 90 minutes the cell wasswitched to dummy and two 100 μl samples were taken, one sample wasstored on ice, the second was incubated at 55° C. in a water bath afteraddition of 2 μl of 10 times concentrated “reannealing buffer” (finalconcentration 20 mM NaCl, 2 mM Tris HCl pH 8.7, 0.2 mM EDTA) for 25hours. The voltage at the working electrode was reversed to +(plus) 1 Vand treatment of the DNA solution in the electrochemical cell proceededfor a further 25 hours. After this second time period 100 μl of the DNAsolution was removed from the cell and stored on ice. Each of the four100 μl samples was mixed with 20 μl of gel loading buffer describedabove and stored on ice until required for electrophoresis.

FIG. 4 is an agarose gel run exactly as described above showing the fourDNA samples; A is the starting intact calf thymus material, B is theelectrically denatured material, C is the electrically denatured andsubsequently electrically renatured material, D is the electricallydenatured material which was subsequently thermally renatured. It can beseen from the gel that both the electrically denatured thermallyrenatured and electrically denatured electrically renatured DNA returnsto the original mobility of the double-stranded starting material.

EXAMPLE 7

This example illustrates that a bacteriophage genome can be electricallydenatured to a single-stranded form in a manner analogous to the thermalmethod. Bacteriophage M13 (M13mp18RF1 Double-stranded form supplied byCP Laboratories, P.O. Box 22, Bishop's Stortford, Herts, UK) wasemployed. M13 is in circular form which can adopt a number of differentcoiled and supercoiled configurations. This leads to a complex set ofbands on the agarose electophoresis gel. Therefore, the gel pattern wassimplified to a single band by subjecting M13 to a restriction digestwith the enzyme BGL I, which has only one restriction site on the M13genome. To one vial of M13 as supplied by CP Laboratories (containing 10μg of M13 in 100 μl of buffer 10 mM Tris HCl pH 7.5 1 mM EDTA) 10 μl of“restriction buffer” was added (0.1 M Tris HCl pH 7.9, 0.01 M magnesiumchloride, 0.05 M sodium chloride) and 6 μl of restriction enzyme (astock at 8,000 U/ml supplied by CP Laboratories No. 143S). The solutionwas incubated at 37° C. in a water bath for 16 hours. To ensure thelinearization was complete, 1 μl of the treated DNA was mixed with 3 μlof distilled water and 1 μl of gel loading buffer (described earlier)was run on a 1% agarose gel. If more than one band was seen afterelectrophoresis at 100 mA for 1 hour and subsequent staining, the DNAwas redigested. Once linearity was determined, the M13 was precipitatedby adding 125 μl of 1 M magnesium chloride, 25 μl of 3 M sodium acetateand 125 μl of absolute ethanol (AnalaR grade from BDH Ltd., Poole, UK).The mixture was frozen in dry ice for 20 minutes for 16 hours and theprecipitate was collected by 15 minute centrifugation after thawing. Thepellet was washed with 0.15 ml of 70% (v/v) ethanol and collected againby centrifugation. Finally, the pellet was dried under vacuum for 15minutes and resuspended in 100 μl of distilled water.

The linearized M13 DNA was then subjected to both thermal and electricaldenaturation. A series of tubes was set up. The series of 6 tubescontained 1 μl M13 DNA with 4 μl of distilled water. One tube was placedon ice, and the other tubes were heated for 2, 4, 6, 8, 10 minutes in aboiling water bath. After a brief period of centrifugation, the tubeswere placed on ice (to prevent renaturation of the thermally denaturedDNA). To each tube 1 μl of gel loading buffer was added. Before beingloaded and subsequently run on a 1% agarose gel at 100 mA for 1 hour,each tube was briefly vortexed.

FIG. 5 shows the results from this thermal denaturation. Track A is thestarting material (0 minutes at 100° C.), Track B is 2 minutes at 100°C., Track C is 6 minutes at 100° C.

It can be clearly seen from the gel that the intact double-stranded M13rapidly disappears (within 4 minutes) from the gel as it denatures tosingle-stranded form which does not bind ethidium bromide stain withhigh efficiency. A faint smear remains on the gel with a higher mobilitythan native double-stranded DNA and this may be the very faintly stainedsingle-stranded material.

For the electrical denaturation experiment, 920 μl of distilled waterwas added to an electrochemical cell as illustrated in FIG. 1. 30 μl ofa 100 mg/ml solution of methyl viologen in distilled water was addedalong with 50 μl of linearized M13 to the electrochemical cell. Thesolution was then mixed gently using the stirring bar. A potential of−(minus) 1.0 V was applied to the working electrode. Samples (50 μl)were taken at timed intervals, and stored on ice until required. The DNAwas precipitated by adding 5 μl of 1 M magnesium chloride, 25 μl of 3 Mammonium acetate and 125 μl of absolute (100%) ethanol to each sample.The precipitate was collected by freezing on dry ice and centrifuging asdescribed above. The pellet was resuspended in 10 μl of distilled waterand subjected to gel electrophoresis.

FIG. 6 shows the time course of the electrical denaturation of M13.Track A is the starting material, Track B is after 5 minutes, Track C isafter 15 minutes. The gel shows the loss of the double-stranded DNAstructure which has disappeared by 15 minutes and the appearance of thefaintly stained single-stranded smear.

EXAMPLE 8

FIG. 7 shows the results from an electrochemical cell polymerase chainreaction (PCR) carried out in the presence of the promoter methylviologen.

30 μl of a methyl viologen stock at 100 mg/ml (so the workingconcentration of methyl viologen was 3 mg/ml) and 50 μl of stock linearM13 were added to 920 μl distilled water (pH not adjusted) in theworking compartment of the cell and gentle stirring achieved by astirring bar. 200 μl of a 3 mg/ml solution of methyl viologen was addedto the reference electrode compartment of the cell. All parts of thisprocedure were carried out at ambient temperature (22° C.). For theinitial cycle of the polymerase chain reaction, a voltage of −(minus) 1V. was applied for 7 minutes and it was observed that the reduced formof the promoter methyl viologen was produced and accumulated such thatall the liquid became blue. Then the potentiostat was switched to dummy.The working/counter electrode was removed from the cell and the solutionleft stirring for 3 minutes and it was observed that the blue colorrapidly disappeared during this period.

Reagents were then added to the cell, with the stirrer bar stillstirring, namely 6 μl of primer (M13 Sequencing primer (−47) 24 mer5′(CGCCAGGGTTTTCCCAGICACGAC)3′ (SEQ ID NO:1) supplied by ALTABiosciences, University of Birmingham, UK, as a 5 μg lyophilized powder)at a final concentration of 78 pmol, 6 μl of reverse primer M13 ReverseSequencing primer (−24)16mer 5′d(AACAGTCATGACCATTG)3′ (SEQ ID NO:2)supplied as a 5 μg lyophilized powder) at a final concentration of 78pmol, 13 μl of deoxynucleotide triphosphate mix (each dNTP present at afinal concentration of 26 μm, supplied by Pharmacia Ltd., MidsummerBoulevard, Milton Keynes, UK), 4 μl buffer mix (at final concentrationof 6.6 mM Tris HCl pH 8, 1 mM MgCl₂) and then 10 μl of Klenow DNApolymerase (supplied by CP Laboratories, as 5000 U/ml, and NorthumbrianBiologicals Ltd., Cramlington, Northumberland, as 5000 U/ml). Excludingthe promoter, the ionic strength was therefore about 20 mM. There wasthen a 7 minute incubation, with gentle stirring for the first 1 minuteand no stirring for the next 6 minutes.

Then the working/counter electrode was replaced and the second cycle ofthe polymerase chain reaction started by −(minus) 1 V. being applied for5 minutes and it was observed that the reduced form of the promotermethyl viologen was produced and accumulated such that all the liquidbecame blue. Then the potentiostat was switched to dummy. Theworking/counter electrode was removed from the cell and the solutionleft stirring for 3 minutes and it was observed that the blue colorrapidly disappeared during this period. Reagents were then added to thecell, with the stirrer bar still stirring, i.e., 13 μl ofdeoxynucleotide triphosphate mix (details as above) and then 2.5 μl ofKlenow DNA polymerase (details as above). There was then a 7 minuteincubation with gentle sting for the first 1 minute and no stirring forthe next 6 minutes.

The second cycle was repeated as third to tenth cycles, omitting theadding of reagents at the end of the tenth cycle.

A sample was taken from the working compartment of the cell (750 μl) andit was split into sub-samples for ease of processing (3×250 μl), then toeach tube was added 50 μl 1 M magnesium chloride, 98 μl 3 M sodiumacetate and 500 μl 100% ethanol. The samples were frozen on dry ice for20 minutes and then after thawing centrifuged for 15 minutes to obtain apellet. The pellet was washed with 250 μl of 70% ethanol and centrifugedas described before. The pellet was dried under vacuum for 15 minutesand then the pellet in each tube was resuspended in 7 μl distilled water(pH not adjusted) and after extensive vortexing and leaving on ice, thecontents of the 3 tubes pooled before running on a gel. 4 μl of gelloading buffer was added to the sample prior to running on a 12%polyacryamide electrophoresis gel (made according to the followingrecipe for three mini gels: 42 ml distilled H₂O, 8 ml TBE buffer (astock of 500 ml distilled water containing 54 g. Trizma Base (SigmaT1503) 25 g. boric acid (Sigma B0252) 20 ml 0.5 M EDTA pH 8 (BDH10093)), 24 ml 40% acrylamide solution (BDH 44354) 6 ml of 2% N′N′methylene bis acrylamide solution (BDH 44355), 400 μl 15% ammoniumpersulphate solution in distilled water (Sigma A7262), 100 μl TEMED (BDH44308)).

In FIG. 7, lane A contains primers, these run faster on the gel than thethermally amplified 119 base pair fragment in lane B. Lanes C and Dcontain 5 μl and 15 μl, respectively, of electrically amplified product.In lanes C and D, high molecular weight M13 DNA is contained in thewell, there is some smearing of DNA in the upper part of the gel, thisis more pronounced in lane D. In both lanes, primers can be seen at thesame mobility as in lane A, however, in lane D extensive “flaring” ofthe primers is observed. The amplified product can be seen in both lanesC and D at the same mobility as the thermally amplified sample in laneB.

EXAMPLE 9

FIG. 8 shows the results from an electrochemical cell polymerase chainreaction (PCR) experiment carried out in the absence of promoter. Themethod used was essentially the same as for Example 8 with promoterdescribed above except that no promoter is added to the cell and alladditions of primer and reverse primer were of 3 μl each (not 6 μl asdescribed above) and the experiment ran for 15 cycles.

In FIG. 8 lane A contains a thermally amplified 119 bp product, lane Bprimers, and lane E stock M13 that is confined to the well due to itshigh molecular weight. Lanes C and D contain the product of electricalamplification. The 119 base pair amplified region is clearly visible atthe same mobility on the gel as the thermally amplified product. Theprimers in lanes C and D run at the same mobility as lane B. Thereduction of the amount of primers added in this experiment incomparison to the experiment illustrated in FIG. 7 reduces the flaringeffect in the gel.

EXAMPLE 10

SK “Blue script” (Stratagene) is a circular 2,964 base pair vector. Itcontains a polylinker region which contains the M13 primer binding sitesbetween which the target region of DNA amplified in Examples 8 and 9 islocated.

Before use in this example, the blue script was linearized usingrestriction enzyme Xmn 1; this cuts at site 2645. Thus, to 80 μmoles ofblue script, approximately 50 μg of DNA, 2.5 μl of −0.13 M −Tris bufferpH 7.9 containing 0.13 M magnesium chloride and 0.3 M sodium chlorideand 8 μl of Xmn 1 restriction enzyme (Stratagene) was added. The mixturewas incubated at 37° C. overnight. After phenol chloroform extractionand ethanol precipitation, the sample was resuspended to 50 μl indistilled water.

To the working compartment of the cell of FIG. 1 was added 1.2 ml ofdistilled water, and 30 μl of 100 mg/ml stock methyl viologen, 200 μl ofthis mixture was pipetted into the reference compartment. 50 μl(approximately 45 μg) of restriction digested SK blue script was addedto the working compartment of the cell and the electrodes were placed intheir respective compartments. Denaturing was conducted for 7 minutes at−1 V., and the working and counter-electrodes were removed from thecell. After 3 minutes stirring, to allow the reoxidation of the methylviologen (a lagphase), the reagents listed below were added. The nucleicacid was allowed to anneal and extend for 5 minutes, the first minutewith stirring and subsequent 4 minutes in the absence of stirring. Thedenaturing, lag phase and reagent addition, annealing and extending stepwere repeated for 10 cycles, but in cycles 2 through 10, thedenaturation step was for 5 minutes.

Addition of reagents:

Cycle 1

4 μl of 1.65 M Tris buffer pH 8.7 containing 330 mM magnesium chloride

13 μl of DATP, dGTP, dTTP and dCTP at 10 mM each in distilled water(CHASE)

10 μl of Klenow DNA polymerase—as in Example B

3 μl of each of concentrated primer (2)

Cycle 2-4 and 6-10 5 μl of CHASE

2.5 μl of Klenow DNA polymerase

After the 10 cycles were completed, the sample was divided into threeand ethanol precipitated, dried and resuspended in 15 μl of distilledwater. The sample was run on a 12% polyacrylamide gel at a constantcurrent of 100 mA for 1 hour. The gel was stained with ethidium bromide.The gel is shown in FIG. 9.

Both the thermally amplified product of M13, a 119 bp band (run as astandard) and the primers can be clearly seen in lanes B and C,respectively. Due to the high concentration of DNA loaded onto lane A,the lane had high background, but a band of greater than 119 bp could beclearly seen. This band is the 200 bp amplified region of SK bluescript.

EXAMPLE 11

The procedure of Example 10 was repeated except that further restrictiondigests were performed to produce 20 μg of linear 3000 base pairbluescript DNA using Xmn 1, and 15 μg of 450 base pair blue script DNAusing Pvu 2 (Stratagene). The electrical PCR amplification process wasperformed in the absence of methyl viologen. A lag phase was notnecessary and was omitted.

The polyacrylamide gel is shown in FIG. 10.

Lane A contains a “ladder marker,” a set of known DNA sizes, which isused to gauge the molecular weight of experimental samples.

Lane B contains the 450 base pair electrically amplified “blue script”DNA. The template 450 base pair band can be clearly seen, as can anamplified band of 200 base pairs.

Lane C contains the 3000 base pair linear electrically amplified “bluescript” DNA. The template DNA is confined to the well (as might beexpected due to its 3000 base pair size) and an amplified band of 200base pairs can clearly be seen.

Many modifications and variations of the invention as specificallydescribed above are possible within the scope of the invention.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 24 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO   (iii) ANTI-SENSE: NO     (vi) ORIGINAL SOURCE:          (A) ORGANISM: none     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #1: CGCCAGGGTT TTCCCAGTCA CGAC           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)   (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:           (A) ORGANISM: none    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:AACAGTCATG ACCATTG              #                   #                  #   17

What is claimed is:
 1. A process for annealing complementary singlestrands of nucleic acid into double-stranded nucleic acid, whichcomprises: applying a negative voltage to said complementary singlestrands of nucleic acid with an electrode and reversing said voltage onsaid electrode to a positive voltage to convert at least a proportion ofsaid complementary single strands of nucleic acid to double-strandednucleic acid.
 2. The process of claim 1, wherein said electrode isimmersed in a solution.
 3. The process of claim 1, wherein said negativevoltage is about −1 volt and said positive voltage is about +1 volt. 4.The process of claim 1, wherein one of said complementary strands ofnucleic acid is immobilized to said electrode.
 5. The process of claim1, wherein said double-stranded nucleic acid is capable of denaturationto a wholly or partially single-stranded form of nucleic acid.
 6. Theprocess of claim 1, wherein at least one of said complementary singlestrands of nucleic acid is bound to a surface comprising said electrode.7. A process for denaturing and annealing nucleic acid, which comprisesthe steps of: (a) applying a negative voltage to a double-strandednucleic acid with an electrode such that at least a proportion of saiddouble-stranded nucleic acid is denatured to wholly or partiallysingle-stranded nucleic acid; and (b) applying a positive voltage tosaid wholly or partially single-stranded nucleic acid with an electrodeto cause said wholly or partially single-stranded nucleic acid to annealwith a complementary nucleic acid.
 8. A process for the annealing ofcomplementary single strands of nucleic acid into double-stranded form,comprising the steps of: applying a positive voltage to saidcomplementary single strands of nucleic acid with an electrode, whereinat least one of said complementary single strands of nucleic acid isbound to an electrode surface; and converting at least a proportion ofsaid complementary single strands to double-stranded nucleic acid. 9.The process of claim 8, wherein said double-stranded nucleic acid iscapable of denaturation into single-stranded form.
 10. A process for theannealing of complementary single strands of nucleic acid intodouble-stranded form, comprising changing a voltage applied to saidcomplementary single strands of nucleic acid from 0 volt to a voltage of+1 volt to convert at least a proportion of said complementary singlestrands of nucleic acid to double-stranded nucleic acid which is capableof denaturation into single stranded form.
 11. A process forelectrically denaturing a plurality of double-stranded nucleic acidmolecules, which comprises: (a) providing a surface having an array ofelectrodes; (b) applying a voltage to each of a plurality ofdouble-stranded nucleic acid molecules with each of a plurality ofelectrodes in the array to convert at least a proportion of each of theplurality of double-stranded nucleic acid molecules to wholly orpartially single stranded form.
 12. A process for electrically annealinga plurality of complementary single strands of nucleic acid intodouble-stranded nucleic acids, which comprises: (a) providing a surfacehaving an array of electrodes; (b) applying a voltage to each of aplurality of complementary single strands of nucleic acid with each of aplurality of electrodes in the array to convert at least a proportion ofeach of the plurality of single strands of nucleic acid to doublestranded nucleic acid.
 13. A process for denaturing and annealing aplurality of nucleic acids, which comprises the steps of: (a) providinga surface having an array of electrodes; (b) applying a negative voltageto each of a plurality of double-stranded nucleic acids with each of aplurality of electrodes in the array such that at least a proportion ofeach of said plurality of double-stranded nucleic acids is denatured towholly or partially single-stranded nucleic acid; and (c) applying apositive voltage to each of a plurality of said wholly or partiallysingle-stranded nucleic acid with each of a plurality of electrodes inthe array to cause each of said plurality of said wholly or partiallysingle-stranded nucleic acid to anneal with a complementary nucleicacid.
 14. A process for the annealing of a plurality of complementarysingle strands of nucleic acid into double-stranded form, comprising thesteps of: (a) providing a surface having an array of electrodes; (b)applying a positive voltage to each of a plurality of said complementarysingle strands of nucleic acid with each of a plurality of theelectrodes in the array, wherein at least one of said complementarysingle strands of nucleic acid is bound to each electrode surface; and(c) converting at least a proportion of said complementary singlestrands to double-stranded nucleic acid.
 15. A process for the annealingof a plurality of complementary single strands of nucleic acid intodouble-stranded form, comprising: (a) providing a surface having anarray of electrodes; (b) changing a voltage applied to each of saidcomplementary single strands of nucleic acid from 0 volt to a voltage of+1 volt with each of a plurality of the electrodes in the array toconvert at least a proportion of said complementary single strands ofnucleic acid to double-stranded nucleic acid which is capable ofdenaturation into single stranded form.